P468: DETECTION AND CHARACTERIZATION OF RESIDUAL LEUKEMIC CELLS IN CHILDHOOD ACUTE MYELOID LEUKEMIA USING TARGETED DEEP SEQUENCING AND SINGLE CELL MULTI-OMICS
Authors: Borhan R Saeed, Anastasia Soboli, Anna Rehammar, Adam Molnar, Erik Delsing Malmberg, Barbara Depreter, Giti Shah Barkhordar, Barbara De Moerloose, Tim Lammens, Lars Palmqvist, Jonas Abrahamsson, Linda Fogelstrand
Journal: HemaSphere (2023)

Background: In children with acute myeloid leukemia (AML), treatment intensification based on measurable residual disease (MRD) detected by multiparameter flow cytometry (MFC) shows promising results. However, a substantial proportion of patients with no detectable MRD by MFC relapse, indicating that more sensitive methods for MRD analysis are needed. Aims: Our aim was to investigate the feasibility of targeted deep sequencing for analysis of MRD during treatment of children with AML. We assessed the sensitivity of targeted deep sequencing as compared to MFC and characterized the genetic and protein signature on the single cell level to understand the nature of residual mutated cells. Methods: Bone marrow samples were collected from children with AML. Leukemia-specific mutations for tracking were identified in diagnostic samples by whole exome sequencing of sorted leukemic cells. Samples from day 22 and before start of second induction and first consolidation courses were assessed with patient-tailored targeted deep sequencing with a limit of detection of 0.02% variant allele frequency. Results were compared to parallel MRD analyses with 8 color MFC with a limit of detection of 0.1% cells, and when applicable RT-qPCR. Single cell multi-omics analysis was performed using the Tapestri platform with 45 primer-barcoded antibodies for cell surface proteins and customized mutation-specific primers for DNA, followed by sequencing. Results: In diagnostic samples, at least one leukemia-specific mutation suitable for tracking was detected in 25/27 patients; most often both clonal and subclonal mutations (average 12 mutations per case). For these children (of which four eventually relapsed), targeted deep sequencing assays to track mutations in samples during treatment were designed for clonal mutations in any gene, and subclonal mutations in cancer-associated genes (in total 74 mutations). When compared to MFC results, targeted deep sequencing was more sensitive; 50% of analyzed samples were positive for at least one mutation with targeted deep sequencing but negative with MFC, 44% were positive with both methods, 6% negative with both methods and none negative with targeted deep sequencing and positive with MFC. In 8 patients assessed for MRD also by RT-qPCR of leukemic transcript, targeted deep sequencing and RT-qPCR showed similar performance. Mutations were detected at the end of induction in patients that eventually relapsed, but also in some patients not experiencing relapse. Therefore, we aimed to characterize residual mutated cells. We used single cell multi-omics analysis on 15 samples from 7 patients (6 diagnostic, 8 during treatment and one relapse) to enable tracking of mutated residual cells and describing protein expression and copy number aberrations. Protein expression signatures were verified using a normal sample with MFC performed in parallel. Our analysis provided detection of MRD on a single cell level, as well as phenotypic characterization of cells harboring leukemia-specific mutations. The detected patterns were patient-specific and included subclonality, as indicated by whole exome and targeted deep sequencing, and the presence of a preleukemic clone in stem/progenitor cells. Summary/Conclusion: Targeted deep sequencing enables a more sensitive detection of MRD than MFC and is applicable in most children with AML. Residual mutated cells during treatment can be relapse-causing but also pre-leukemic. Further studies are needed to address the prognostic value of highly sensitive molecular MRD in childhood AML. Keywords: OMICS, Quantitative molecular analysis, Acute myeloid leukemia, MRD